In the fields of medical, dietary, environmental and chemical sciences, there is an increasing need for the selective separation of specific substances from complex mixtures of related substances. The goals vary; it may be the preparative isolation of one or more specific compounds, or measurement of their concentration, or the selective removal of a target compound from a multi-component mixture.
Riboflavin (vitamin B2), a water soluble vitamin, is essential for human diet being a major, component of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the co-enzymes responsible for redox reactions. It is found in liver (3.5 mg/100 g), cheese (0.5 mg/100 g), milk (0.15 mg/100 g) and in beer (ca. 200 μg/L). While riboflavin is relatively stable towards heat and acidic pH, it decomposes in the presence of alkalis and light to lumiflavin, a stronger oxidative agent which contributes to the decomposition of vitamin C. The photoreduction of riboflavin is responsible for the break-down of the bitter iso-alpha acids in the presence of a sulphur source, which leads to the well-known “sun-struck” flavour of white wine, champagne, milk and beer which have been exposed to sunlight. In order to prevent the development of such undesired flavours, these products are preferably stored in bottles that are dark and non-transparent to light in the case of beer and wine, or, in plastic/paper packaging, in the case of milk. These measures would not be necessary for products lacking riboflavin.
Methods to selectively remove riboflavin from complicated matrices without otherwise affecting their composition are therefore of interest. The use of riboflavin binding protein in an affinity separation mode was suggested by M. G. Duyvis, et al., J. Agric. Food. Chem. 2002, Vol. 50, pp. 1548-1552, and by C. Laane, et al., J. Inst. Brew. 1999, Vol. 105, p. 392. However, the procedures suggested were strongly limited by issues related to stability and cost, which severely reduced the feasibility of these procedures.
Instead the use of stable polymeric materials capable of removing riboflavin from complicated matrices without otherwise affecting their composition may be more useful in this regard. Materials able to show such properties may thus find important applications in enhancing the stability and quality of relevant food and drink products. Moreover, such materials may be used to enrich and isolate flavins from complicated matrices prior to analytical quantification, or, as recognition elements in chemical sensors.
One such class of materials is the class known as “molecularly imprinted polymers (MIPs)”, which may be prepared using the technique of molecular imprinting.
The most widely used protocol for the preparation of molecularly imprinted polymers (MIPs) (hereafter referred to as “the conventional technique or procedure”) entails the following key steps:
(i) The target or template molecule, or else a structural analogue of it (“T”), is allowed to contact and interact with, the selected functional monomer (“M”) in an aprotic solvent of low polarity, to form template-monomer assemblies which are noncovalently associated,
(ii) The template-monomer assemblies are copolymerised with a cross-linking agent or monomer (“M-M”) resulting in a cross-linked porous network polymer (also known as polymer matrix),
(iii) The target or template (“T”) is extracted from the polymer matrix, leaving the resulting MIP possessing the corresponding binding sites.
These binding sites on the MIP are capable of selectively re-binding the corresponding target or template molecule, or a close structural analogue, with high affinity and selectivity. Often the binding selectivities of these sites can be compared with the antibody-antigen complementarity.
In the conventional technique described above, the MIP is typically crushed and sieved prior to step (iii) (extraction of the template), to obtain a desired size fraction of particulate material. These can then be packed into a chromatographic column and used for chromatographic separation of the template from other components of a mixture with similar structure or functionality. Analytical as well as preparative applications are possible. In preparative applications, the purpose may be to isolate or to remove a particular compound. This may be performed, for example, through an affinity chromatographic procedure where either pH, ion strength or solvent gradients, or a combination of said parameters, may be used in order to control the strength of interaction with the stationary phase. The crude mixture is typically allowed to pass through a packed bed of the MIP whereby the compound to be removed or isolated is selectively retained on the MIP. Subsequently, the compound is released from the MIP in a regeneration step. After a conditioning step, the MIP is ready for reuse.
The use of MIPs in membrane format is yet an alternative, which may offer benefits by allowing faster separations and the possibility for continuous production of pure compounds.
Alternatively, separation may be performed in a so-called “batch format” where the MIP is suspended in the crude mixture for a time period considered sufficient for selective adsorption of the compound to occur. The regeneration can thereafter be performed as described above.
MIPs show promise in chiral separations of, for example, amino acid derivatives, peptides, phosphonates, aminoalcohols including beta-blocking compounds, and a number of chiral drugs.
Furthermore, promising developments involving MIPs are seen in affinity chromatography (See for ex., Y. Yu, et al., Biotechnology and Bioengineering 2002, Vol. 79, pp. 23-28), in chemical sensing (See for ex., K. Haupt, et al., Chem. Rev. 2000, Vol. 100, pp. 2495-2504) and as substitutes for antibodies in immunoassays of small target analytes (See for ex., L. Ye, et al., J. Am. Chem. Soc. 2001, Vol. 123, pp. 2901-2902). The patent literature reveals that the materials may find commercial use in all of these mentioned applications.
The polymerization process in the conventional procedure is performed in the presence of a pore-forming solvent called a porogen. In order to stabilize electrostatic interactions between the functional monomers and the template, the porogen selected is often an aprotic solvent possessing low to moderate polarity. The majority of templates used at the present time exhibit moderate to high solubility in such solvents (hereafter known as “conventional solvents”), and these or their structural analogues can therefore be imprinted using the conventional procedure.
On the other hand, the conventional procedure described above is not possible for hydrophilic targets or templates, which includes the majority of biologically interesting molecules. For this class of target or template molecules, the present imprinting techniques are associated with two major problems.
The first problem relates to the limited solubility of such targets in the conventional solvents.
Riboflavin, or analogues thereof (for example, FAD, FMN), belongs to the class of water-soluble vitamins and exhibits minimal to zero solubility in the low to non-polar, organic solvents typically used in the technique of molecular imprinting. It is therefore not possible to use riboflavin itself as a template in the conventional MIP synthesis techniques.
The second problem relates to the occurrence of non-specific hydrophobic binding when MIPs produced using conventional techniques are used as sorbents in water. Due to the hydrophobic nature of the matrix monomer, most targets adsorb nonspecifically to the polymer surface when the materials are used in pure aqueous media.
Measures to suppress this non-specific binding must thus be found. In order to obtain MIP sorbents capable of strongly and specifically adsorbing or binding hydrophilic biomolecules, e.g. riboflavin, or analogues thereof, from water rich media, approaches which lead to (1) imprinted sites capable of binding the target or template molecule in water and (2) suppression of non-specific binding, must be found.
To achieve imprinted sites capable of binding the template in water, a riboflavin analogue satisfying the following criteria, i.e., (a) is soluble in conventional solvents, (b) is stable under polymerisation conditions, (c) has close structural and shape analogy with riboflavin resulting in an imprinted site capable of accommodating riboflavin in aqueous media, may be used as a template.